Pregnancy-associated Plasma Protein-A (PAPP-A)
PAPP-A was first isolated in 1974 from pregnancy serum along with other proteins believed to be of placental origin (Lin et al., 1974, Am J Obstet Gynecol 118, 223–36). The concentration in serum reaches about 50 mg/liter at the end of pregnancy (Folkamen et al., 1981, Am J Obstet Gynecol 139, 910–4; Oxvig et al., 1995, J Biol Chem 270, 13645–51). PAPP-A was originally characterized as a high molecular weight homotetramer (Bischof, 1979, Arch Gynecol 227, 315–26; Lin et al., 1974, Am J Obstet Gynecol 118, 223–36; Sinosich, 1990, Eleotrophoresis 11, 70–8), but it has now been demonstrated that PAPP-A primarily exists in pregnancy serum and plasma as a covalent, heterotetrameric 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP (Oxvig et al., 1993, J Biol Chem 268, 12243–6). Only about 1% of PAPP-A in pregnancy serum and plasma is present as a homodimer, as recently demonstrated (Overgaard et al., 2000, J Biol Chem). The existence of the PAPP-A/proMBP complex was revealed, in part, by the isolation of a PAPP-A and a proMBP peptide, linked together by a disulfide bond, from a digest of purified PAPP-A/proMBP (Oxvig et al., 1993, J Biol Chem 268, 12243–6).
The subunits of the PAPP-A/proMBP complex can be irreversibly separated by reduction of disulfide bonds and denaturation (Oxvig et al., 1993, J Biol Chem 268, 12243–6). In reducing SDS-PAGE, the PAPP-A subunit has an apparent molecular weight of 200 kDa (Oxvig et al., 1994, Biochim Biophys Acta 1201, 415–23), and its 1547-residue sequence is known from cloned cDNA (Kristensen et al., 1994, Biochemistry 33, 1592–8) PAPP-A is synthesized as a pre-pro-protein (preproPAPP-A), including a 80-residue prepro-piece(Haaning et al., 1996, Eur J Biochem 237, 159–63). No proteins with global homology to PAPP-A has been reported in the literature, but PAPP-A contains sequence motifs, including an elongated zinc binding motif (HEXXHXXGXXH) (SEQ ID NO:26) at position 482–492 (numbering according to Kristensen et al., 1994, Biochemistry 33, 1592–8). This motif and a structurally important methionine residue, also thought to reside in PAPP-A at position 556, are strictly conserved within the metzincins, a superfamily of zinc peptidases: astacins, adamalysins (or reprolysins), serralysins and matrixins (matrix metalloproteinases or MMP's) (Bode et al., 1993, FEBS Lett 331, 134–40; Stocker et al., 1995, Protein Sci 4, 823–40).
The proMBP subunit has a calculated peptide mass of 23 kDa (Barker et al., 1988, J Exp Med 168, 1493–8; McGrogan et al., 1988, J Exp Med 168, 2295–308). In SDS-PAGE, however, proMBP migrates as a smear of 5–90 kDa that is not visible in Coomassie-stained gels (Oxvig et al., 1993, J Biol Chem 268, 12243–6), probably due to its strong and unusual glycosylation (Oxvig et al. 1994, Biochem Mol Biol Int 33, 329–36, Oxvig et al. 1994, Biochim Biophys Acta 1201, 415–23). PAPP-A and proMBP are both produced in the placenta during pregnancy, but mainly in different cell types as shown by in situ hybridization (Bonno et al., 1994, Lab Invest 71, 560–6). Analyses by RT-PCR revealed that both PAPP-A and proMBP mRNA are present in several reproductive and nonreproductive tissues, although the levels are lower than in the placenta (Overgaard et al., 1999, Biol Reprod 61, 1083–9).
Clinical Use of PAPP-A
Clinically, depressed serum levels of PAPP-A are increasingly being used as a predictor of Down's syndrome pregnancies (Brambati et al., 1993, Br J Obstet Gynaecol 100, 324–6; Haddow et al., 1998, N Engl J Med 338, 955–61; Wald et al., 1992, Bmi 305, 28: Wald et al., 1999, N Engl J Med 341, 461–7), and it has been shown that PAPP-A serum levels are also depressed in other fetal abnormalities (Biagiotti et al., 1998, Prenat Diagn 18, 907–13; Spencer et al., 2000, Prenat Diagn 20, 411–6; Westergaard et al., 1983, Prenat Diagn 3, 225–32).
Further, the synthesis of PAPP-A in smooth muscle cells of the coronary artery following angioplasty is increased (Bayes-Genis et al., 2000, Arteriosclor Thromb Vaso Biol, in press), which is currently being evaluated for potential clinical value. Data show that measurements of proMBP in pregnancy serum also have a diagnostic value (Christiansen et al., 1999, Prenat Diagn 19, 905–10).
Proteolytic Activity of PAPP-A: Cleavage of IGFBP-4
Only recently, the putative metalloproteinese activity of PAPP-A has been experimentally confirmed (Lawrence et al., 1999, Proc Natl Acad Sci USA 96, 3149–53). PAPP-A was partially purified from human fibroblast-conditioned medium (HFCM) and shown to be responsible for the proteolytic activity of HFCM against insulin-like growth factor binding protein (IGFBP)-4. IGFBP's, of which six have been described, are important modulators of IGF-I and -II activity (Fowlkes, 1997, Trends Endocrinol Metab 8, 299–306, Rajaram et al., 1997, Endocr Rev 18, 801–31).
IGF-I and -II are essential polypeptides with potent anabolic and mitogenic actions both in vivo and in vitro. IGF bound to IGFBP-4 cannot interact with its receptor, but bioactive IGF is released once the binding protein is cleaved. Interestingly, cleavage of IGFBP-4 by PAPP-A strictly requires the presence of IGF (Conover et al., 1993, J Clin Invest 91, 1129–37; Lawrence et al., 1999, Pro Natl Acad USA 96, 3149–53). PAPP-A secretion has also been demonstrated from osteoblasts and marrow stromal cells (Lawrence et al., 1999, Proc Natl Aced Sci USA 96, 3149–53), from granulosa cells (Conover at al., 1999, J Clin Endocrinol Metab 84, 4742–5), and from vascular smooth muscle cells (Bayes-Genis et al., 2000, Arterloscler Thromb Vasc Biol. in press), all of which have known IGF-dependent IGFBP-4 proteinase activity.
IGFBP-5
Like IGFBP-4. IGFBP-5 cleavage has been widely reposed to occur by unidentified proteinases is a number of tissues and conditioned media (Hwa at al., 1999, Endocr Rev 20, 761–87).